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1
Björk, I., E. Pol, E. Raub-Segall, M. Abrahamson, A. D. Rowan, and J. S. Mort. "Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes." Biochemical Journal 299, no. 1 (April 1, 1994): 219–25. http://dx.doi.org/10.1042/bj2990219.
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The importance of the N-terminal region of human cystatin C or chicken cystatin for the kinetics of interactions of the inhibitors with four cysteine proteinases was characterized. The association rate constants for the binding of recombinant human cystatin C to papain, ficin, actinidin and recombinant rat cathepsin B were 1.1 x 10(7), 7.0 x 10(6), 2.4 x 10(6) and 1.4 x 10(6) M-1.s-1, whereas the corresponding dissociation rate constants were 1.3 x 10(-7), 9.2 x 10(-6), 4.6 x 10(-2) and 3.5 x 10(-4) s-1. N-Terminal truncation of the first ten residues of the inhibitor negligibly affected the association rate constant with papain or ficin, but increased the dissociation rate constant approx. 3 x 10(4)- to 2 x 10(6)-fold. In contrast, such truncation decreased the association rate constant with cathepsin B approx. 60-fold, while minimally affecting the dissociation rate constant. With actinidin, the truncated cystatin C had both an approx. 15-fold lower association rate constant and an approx. 15-fold higher dissociation rate constant than the intact inhibitor. Similar results were obtained for intact and N-terminally truncated chicken cystatin. The decreased affinity of human cystatin C or chicken cystatin for cysteine proteinases after removal of the N-terminal region is thus due to either a decreased association rate constant or an increased dissociation rate constant, or both, depending on the enzyme. This behaviour indicates that the contribution of the N-terminal segment of the two inhibitors to the interaction mechanism varies with the target proteinase as a result of structural differences in the active-site region of the enzyme.
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Hofsteenge, J., H. Taguchi, and S. R. Stone. "Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors." Biochemical Journal 237, no. 1 (July 1, 1986): 243–51. http://dx.doi.org/10.1042/bj2370243.
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Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.
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Boudier, C., and J. G. Bieth. "Oxidized mucus proteinase inhibitor: a fairly potent neutrophil elastase inhibitor." Biochemical Journal 303, no. 1 (October 1, 1994): 61–68. http://dx.doi.org/10.1042/bj3030061.
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N-chlorosuccinimide oxidizes one of the methionine residues of mucus proteinase inhibitor with a second-order rate constant of 1.5 M-1.s-1. Cyanogen bromide cleavage and NH2-terminal sequencing show that the modified residue is methionine-73, the P′1 component of the inhibitor's active centre. Oxidation of the inhibitor decreases its neutrophil elastase inhibitory capacity but does not fully abolish it. The kinetic parameters describing the elastase-oxidized inhibitor interaction are: association rate constant kass. = 2.6 x 10(5) M-1.s-1, dissociation rate constant kdiss. = 2.9 x 10(-3) s-1 and equilibrium dissociation constant Ki = 1.1 x 10(-8) M. Comparison with the native inhibitor indicates that oxidation decreases kass. by a factor of 18.8 and increases kdiss. by a factor of 6.4, and therefore leads to a 120-fold increase in Ki. Yet, the oxidized inhibitor may still act as a potent elastase inhibitor in the upper respiratory tract where its concentration is 500-fold higher than Ki, i.e. where the elastase inhibition is pseudo-irreversible. Experiments in vitro with fibrous human lung elastin, the most important natural substrate of elastase, support this view: 1.35 microM elastase is fully inhibited by 5-6 microM oxidized inhibitor whether the enzyme-inhibitor complex is formed in the presence or absence of elastin and whether elastase is pre-adsorbed on elastin or not.
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Brown, Nicholas G., Dar-Chone Chow, and Timothy Palzkill. "BLIP-II Is a Highly Potent Inhibitor of Klebsiella pneumoniae Carbapenemase (KPC-2)." Antimicrobial Agents and Chemotherapy 57, no. 7 (April 15, 2013): 3398–401. http://dx.doi.org/10.1128/aac.00215-13.
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ABSTRACTβ-Lactamase inhibitory protein II (BLIP-II) is a potent inhibitor of class A β-lactamases. KPC-2 is a class A β-lactamase that is capable of hydrolyzing carbapenems and has become a widespread source of resistance to these drugs for Gram-negative bacteria. Determination of association and dissociation rate constants for binding between BLIP-II and KPC-2 reveals a very tight interaction with a calculated (koff/kon) equilibrium dissociation constant of 76 fM (76 × 10−15M).
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LOHSE, Anders, Tore HARDLEI, Astrid JENSEN, Igor W. PLESNER, and Mikael BOLS. "Investigation of the slow inhibition of almond β-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the ‘direct binding’ model." Biochemical Journal 349, no. 1 (June 26, 2000): 211–15. http://dx.doi.org/10.1042/bj3490211.
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(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This ‘slow inhibition’ process was investigated in the inhibition of almond β-glucosidase and yeast isomaltase by inhibitor 1 and analogues. Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations. A number of hydrazine-containing analogues of inhibitor 1 also inhibited β-glucosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited β-glucosidase slowly. Inhibitor 1 and related inhibitors were found to leave almond β-glucosidase with almost identical rate constants, so that the difference in Ki values depended almost entirely on changes in the binding rate constant, kon. The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor. The values of the rate constants were obtained at 25 °C and at pH 6.8.
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Lindahl, P., E. Raub-Segall, S. T. Olson, and I. Björk. "Papain labelled with fluorescent thiol-specific reagents as a probe for characterization of interactions between cysteine proteinases and their protein inhibitors by competitive titrations." Biochemical Journal 276, no. 2 (June 1, 1991): 387–94. http://dx.doi.org/10.1042/bj2760387.
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Papain was labelled by attachment of the fluorescent groups 2-(4′-acetamidoanilino)naphthalene-6-sulphonic acid (AANS) or N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid (AEDANS) to the active-site cysteine residue, with the aim of using the labelled papains as probes in competitive titrations of unlabelled cysteine proteinases with their inhibitors. The interaction between the labelled papains and cystatins was accompanied by an increase in fluorescence emission of up to 38-fold for AANS-papain and approximately 3.5-fold for AEDANS-papain. Fluorescence titrations gave dissociation equilibrium constants of 3.1 and 0.6 microM for the binding of chicken cystatin and recombinant human cystatin C respectively to AANS-papain and of 11.9 microM for the binding of chicken cystatin to AEDANS-papain. The kinetics of interaction of chicken cystatin with AANS-papain showed an unusual biphasic dependence of the observed pseudo-first-order rate constant on inhibitor concentration, consistent with the reaction occurring via both pathways of a general two-step binding mechanism. AANS-papain was selected as the most suitable probe for competitive titrations of unlabelled active or inactivated cysteine proteinases with inhibitors. This technique, which provides stoichiometries and dissociation constants for the interaction between unlabelled enzyme and inhibitor, allows monitoring of the interactions by a large fluorescent signal in a wavelength region where the interacting proteins do not contribute to the observed fluorescence. Such competitive titrations of active papain or actinidin with chicken cystatin or recombinant human cystatin C all gave inhibitor/enzyme stoichiometries of close to 1.0. A dissociation constant of 1.8 microM for the reaction of chicken cystatin with a papain derivative, S-[N-(3-carboxypropyl)succinimidyl]-papain, was also determined by the same technique. These results show the usefulness of the fluorescent papains for the characterization of interactions between cysteine-proteinase inhibitors and their target enzymes.
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Lindhout, T., G. Willems, R. Blezer, and H. C. Hemker. "Kinetics of the inhibition of human factor Xa by full-length and truncated recombinant tissue factor pathway inhibitor." Biochemical Journal 297, no. 1 (January 1, 1994): 131–36. http://dx.doi.org/10.1042/bj2970131.
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The inhibition equilibrium and kinetics of association and dissociation of the binding of three types of recombinant tissue factor pathway inhibitor (TFPI), namely full-length TFPI, C-terminal-truncated TFPI, and TFPI without the third Kunitz domain (TFPI1-161), to factor Xa have been measured. Formation and dissociation of the complexes were monitored by continuous measurement of the changes in the rate of hydrolysis of a peptidyl-p-nitroanilide substrate. Progress curves of product formation were fitted to a set of equations describing a one-step bimolecular inhibitory reaction in the presence of a competing substrate. For full-length TFPI the rate constants of association (kon) and dissociation (koff) were (5.1 +/- 0.7) x 10(6) M-1.s-1 and (2.6 +/- 0.9) x 10(-4)s-1 respectively. Thus, although the inhibition constant (50 pM) is far below the plasma concentration (2.5 nM) of TFPI, the half-time for transition to equilibrium in plasma is rather long (66s). The truncated forms of TFPI differ in that they have a 4-fold lower kon value but a similar dissociation rate constant. Therefore the inhibition constant, Ki, is 4-fold higher (0.2 nM) and the half-time to achieve equilibrium is prolonged to 250 s. The kon values of full-length and C-terminal-truncated TFPI, but not that of TFPI1-161, were found to decrease with increasing ionic strength.
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Ahmad, Suhail, R. K. Bhatnagar, and T. A. Venkitasubramanian. "Ornithine transcarbamylase from Mycobacterium smegmatis ATCC 14468: purification, properties, and reaction mechanism." Biochemistry and Cell Biology 64, no. 12 (December 1, 1986): 1349–55. http://dx.doi.org/10.1139/o86-177.
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Ornithine transcarbamylase (EC 2.1.3.3) has been purified 980-fold from Mycobacterium smegmatis and has a molecular weight of 116 000. Initial velocity determinations indicated that the reaction proceeds via a sequential kinetic mechanism. The limiting Michaelis constants for carbamyl phosphate (KmA) and ornithine (KmB) and the dissociation constant for carbamyl phospate (Kia) were found to be 0.20, 0.25, and 0.07 mM, respectively. Ornithine at higher concentrations acted as an uncompetitive inhibitor when carbamyl phosphate was the variable substrate. Phosphate was a competitive inhibitor with carbamyl phosphate as variable substrate and showed noncompetitive or mixed type inhibition when ornithine was the variable substrate. Norvaline acted as a competitive inhibitor with ornithine as variable substrate and as an uncompetitive inhibitor when carbamyl phophate was the variable substrate. Such inhibitory patterns are characteristic of reactions that proceed via sequential ordered mechanisms. Although the enzyme activity was strongly inhibited by arginine, several arginine analogs had no effect on the enzyme activity. The results suggest that, even though the enzyme from M. smegmatis is unique in the sense that it is feedback inhibited by arginine, the reaction mechanism is similar to the ornithine transcarbamylase isolated from other microorganisms.
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Jin, Byung-Ju, Jay R. Thiagarajah, and A. S. Verkman. "Convective washout reduces the antidiarrheal efficacy of enterocyte surface–targeted antisecretory drugs." Journal of General Physiology 141, no. 2 (January 28, 2013): 261–72. http://dx.doi.org/10.1085/jgp.201210885.
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Secretory diarrheas such as cholera are a major cause of morbidity and mortality in developing countries. We previously introduced the concept of antisecretory therapy for diarrhea using chloride channel inhibitors targeting the cystic fibrosis transmembrane conductance regulator channel pore on the extracellular surface of enterocytes. However, a concern with this strategy is that rapid fluid secretion could cause convective drug washout that would limit the efficacy of extracellularly targeted inhibitors. Here, we developed a convection–diffusion model of washout in an anatomically accurate three-dimensional model of human intestine comprising cylindrical crypts and villi secreting fluid into a central lumen. Input parameters included initial lumen flow and inhibitor concentration, inhibitor dissociation constant (Kd), crypt/villus secretion, and inhibitor diffusion. We modeled both membrane-impermeant and permeable inhibitors. The model predicted greatly reduced inhibitor efficacy for high crypt fluid secretion as occurs in cholera. We conclude that the antisecretory efficacy of an orally administered membrane-impermeant, surface-targeted inhibitor requires both (a) high inhibitor affinity (low nanomolar Kd) to obtain sufficiently high luminal inhibitor concentration (>100-fold Kd), and (b) sustained high luminal inhibitor concentration or slow inhibitor dissociation compared with oral administration frequency. Efficacy of a surface-targeted permeable inhibitor delivered from the blood requires high inhibitor permeability and blood concentration (relative to Kd).
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Bernal, A. López, S. Buckley, C. M. P. Rees, and J. M. Marshall. "Meclofenamate inhibits prostaglandin E binding and adenylyl cyclase activation in human myometrium." Journal of Endocrinology 129, no. 3 (June 1991): 439–45. http://dx.doi.org/10.1677/joe.0.1290439.
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ABSTRACT The effect of sodium meclofenamate on the binding of [3H]prostaglandin E2 ([3H]PGE2) to membranes from human myometrium was investigated. Meclofenamate inhibited the binding of [3H]PGE2 to high-affinity (dissociation constant 1·5 nmol/l) sites in a reversible dose-dependent manner (inhibition constant 11 μmol/l). The mechanism of inhibition was mainly competitive, but at high doses of meclofenamate (≥ 100 μmol/l) there was loss of PGE receptor sites. Of several PG synthesis inhibitors tested, only meclofenamate and, to a lesser extent, mefenamic acid had a significant inhibitory effect. PGE2 stimulated cyclic AMP generation in slices of human myometrium and this was inhibited by meclofenamate in a dose-dependent manner (50% inhibition occurred at 9 μmol/l). Again, this effect was specific for meclofenamate and fitted a competitive mechanism at doses in the range 1–10 μmol/l and a non-competitive mechanism at higher doses. The data show that meclofenamate, in addition to its traditional role as a PG synthesis inhibitor, affects directly PGE receptor binding and activation. Journal of Endocrinology (1991) 129, 439–445
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VENÄLÄINEN, Jarkko I., Risto O. JUVONEN, J. Arturo GARCIA-HORSMAN, Erik A. A. WALLÉN, Johannes A. M. CHRISTIAANS, Elina M. JARHO, Jukka GYNTHER, and Pekka T. MÄNNISTÖ. "Slow-binding inhibitors of prolyl oligopeptidase with different functional groups at the P1 site." Biochemical Journal 382, no. 3 (September 7, 2004): 1003–8. http://dx.doi.org/10.1042/bj20040992.
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POP (prolyl oligopeptidase) specifically hydrolyses a number of small proline-containing peptides at the carboxy end of the proline residue and POP inhibitors have been shown to have cognition-enhancing properties. It has been noted that certain functional groups at the P1 site of the inhibitor, which correspond to the substrate residue on the N-terminal side of the bond to be cleaved, increase the inhibitory potency. However, detailed mechanistic and kinetic analysis of the inhibition has not been studied. In the present study, we examined the effect of different functional groups at the P1 site of the parent inhibitor isophthalic acid bis-(L-prolylpyrrolidine) amide on the binding kinetics to POP. Addition of CHO, CN or COCH2OH groups to the P1 site increased the inhibitory potency by two orders of magnitude (Ki=11.8–0.1 nM) and caused a clear slow-binding inhibition. The inhibitor containing a CHO group had the lowest association rate constant, kon=(2.43±0.12)×105 M−1·s−1, whereas the inhibitor with a CN group exhibited the fastest binding, kon=(12.0±0.08)×105 M−1·s−1. In addition, the dissociation rate was found to be crucially dependent on the type of the functional group. Compounds with COCH2OH and CHO groups had much longer half-lives of dissociation (over 5 h) compared with the compound with the CN group (25 min), although the Ki values of the compounds were relatively similar. A possibility to optimize the duration of inhibition by changing the functional group at the P1 site is important when planning therapeutically useful POP inhibitors.
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Levesque, Marc C., Dipak K. Ghosh, Bethany E. Beasley, Youwei Chen, Alicia D. Volkheimer, Charles W. O’Loughlin, Jon P. Gockerman, Joseph O. Moore, and J. Brice Weinberg. "Induction of Chronic Lymphocytic Leukemia (CLL) Apoptosis by Nitric Oxide Synthase (NOS) Inhibitors: Drug Efficacy Correlates with Lipid Solubility and NOS1 Dissociation Constant." Blood 106, no. 11 (November 16, 2005): 5044. http://dx.doi.org/10.1182/blood.v106.11.5044.5044.
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Abstract The viability of CLL cells may be dependent on the autocrine production of nitric oxide because nitric oxide synthase (NOS) inhibitors induce CLL cell apoptosis and CLL cells express inducible NOS (NOS2). Our previous study indicated that the non-specific NOS inhibitor NMMA induced CLL cell apoptosis but only at high concentrations (> 1 mM) (Levesque et al., Leukemia17:442, 2003). Therefore, we performed the current study to identify NOS inhibitors that induce CLL cell apoptosis at lower concentrations and to understand factors that promote NOS inhibitor-induced CLL cell toxicity. We isolated and enriched CLL cells from the blood of CLL patients and cultured the CLL cells in media containing various concentrations of 21 different NOS inhibitors. We determined CLL cell viability following culture with each NOS inhibitor. We found that NOS inhibitors with specificity for neuronal NOS (NOS1) induced CLL cell death at concentrations lower than non-specific NOS inhibitors and lower than inducible NOS (NOS2) specific inhibitors. There was a weak correlation (r2 = 0.29, p = 0.1608) of the NOS1 (but not NOS2) half-maximal inhibitory concentration (IC50) of each NOS inhibitor for purified recombinant NOS and its ability to induce CLL cell death. We confirmed the specificity of the NOS inhibitors by inhibition of purified recombinant NOS1 and NOS2 enzyme activity, and we confirmed that NOS1 specific inhibitors induced CLL cell death by apoptosis. Because there was only a weak correlation of the NOS1 IC50 with NOS inhibitor induced CLL cell death, we considered whether other factors such as the Kd and hydrophobicity of each compound correlated with CLL cell death. We found that there was a direct correlation between the NOS1 (but not NOS2) dissociation constant (Kd) of NOS inhibitors and CLL cell death (r2 = 0.77, p = 0.0041) and a direct correlation of the partitioning coefficient (a measure of hydrophobicity) of each NOS inhibitor and its ability to induce CLL cell death (r2 = 0.68, p < 0.0001). Therefore, NOS inhibitors that bound tightly to NOS1 and were hydrophobic induced CLL cell death at lower concentrations. There was variable expression of CLL cell NOS1 mRNA (6 of 28 samples positive) and we were unable to demonstrate CLL cell expression of NOS1 protein by immunoblotting. This suggests that if NOS1 is present in CLL cells, it exists at very low levels. Taken together, we believe that low level NO production promotes CLL cell viability and that inhibition of CLL NOS induces CLL cell apoptosis. Importantly, our studies provide direction for the rational design and selection of NOS inhibitors that may be useful as CLL therapeutics.
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Waley, S. G. "The kinetics of slow-binding and slow, tight-binding inhibition: the effects of substrate depletion." Biochemical Journal 294, no. 1 (August 15, 1993): 195–200. http://dx.doi.org/10.1042/bj2940195.
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Inhibitors with dissociation constants in the micromolar to nanomolar range are important, but hard to characterize kinetically, especially when the substrate concentration in the assay is less than Km. When inhibition increases during the course of the assay (slow-binding inhibition) the concentration of substrate may decrease appreciably. Methods that take substrate depletion into account are described for analysing experiments in which the initial substrate concentration is below Km. Fitting progress curves gives the rate constants for the second (slow) step in a two-step mechanism. An approximate value for the overall dissociation constant may be determined from measurements of rates when the reaction is treated as a first-order process. When the concentrations of inhibitor and enzyme are comparable numerical methods are required. Procedures, suitable for implementation on a microcomputer, for the solution of the differential equations and the fitting of progress curves are described.
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Mende, Katrin, Goetz Nowak, and Mercedes López. "Improvement of the specificity of dipetarudin by site directed mutagenesis." Thrombosis and Haemostasis 93, no. 03 (2005): 430–36. http://dx.doi.org/10.1160/th04-08-0480.
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SummaryProtease specificity is crucial to the design of thrombin inhibitors as inhibition of other physiologically relevant serine-proteases can compromise their clinical use. Dipetarudin, a potent thrombin inhibitor, also inhibits trypsin and plasmin. Due to the specificity of an inhibitor being influenced by the amino acid residue at the P1 position, we replaced the Arg10 at P1 position of dipetarudin by a histidine, which is the P1 residue of rhodniin, a very specific thrombin inhibitor. The amino acid replacement was carried out by site directed mutagenesis. The mutant, dipetarudin R10H, showed a loss of plasmin and trypsin inhibitory activities present in its wild-type counterpart and a 3-fold higher dissociation constant for thrombin than dipetarudin. However, compared to dipetarudin and r-hirudin, dipetarudin R10H showed similar activity in coagulation screening assays such as activated partial thromboplastin time (aPTT), prothrombin time (PT), ecarin clotting time (ECT) and ecarin chromogenic assay (ECA).
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Ramić, Alma, Ana Matošević, Barbara Debanić, Ana Mikelić, Ines Primožič, Anita Bosak, and Tomica Hrenar. "Synthesis, Biological Evaluation and Machine Learning Prediction Model for Fluorinated Cinchona Alkaloid-Based Derivatives as Cholinesterase Inhibitors." Pharmaceuticals 15, no. 10 (September 30, 2022): 1214. http://dx.doi.org/10.3390/ph15101214.
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A series of 46 Cinchona alkaloid derivatives that differ in positions of fluorine atom(s) in the molecule were synthesized and tested as human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. All tested compounds reversibly inhibited AChE and BChE in the nanomolar to micromolar range; for AChE, the determined enzyme-inhibitor dissociation constants (Ki) ranged from 3.9–80 µM, and 0.075–19 µM for BChE. The most potent AChE inhibitor was N-(para-fluorobenzyl)cinchoninium bromide, while N-(meta-fluorobenzyl)cinchonidinium bromide was the most potent BChE inhibitor with Ki constant in the nanomolar range. Generally, compounds were non-selective or BChE selective cholinesterase inhibitors, where N-(meta-fluorobenzyl)cinchonidinium bromide was the most selective showing 533 times higher preference for BChE. In silico study revealed that twenty-six compounds should be able to cross the blood-brain barrier by passive transport. An extensive machine learning procedure was utilized for the creation of multivariate linear regression models of AChE and BChE inhibition. The best possible models with predicted R2 (CD-derivatives) of 0.9932 and R2(CN-derivatives) of 0.9879 were calculated and cross-validated. From these data, a smart guided search for new potential leads can be performed. These results pointed out that quaternary Cinchona alkaloids are the promising structural base for further development as selective BChE inhibitors which can be used in the central nervous system.
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Martin, G. E. M., N. G. Rutherford, P. J. F. Henderson, and A. R. Walmsley. "Kinetics and thermodynamics of the binding of forskolin to the galactose-H+ transport protein, GalP, of Escherichia coli." Biochemical Journal 308, no. 1 (May 15, 1995): 261–68. http://dx.doi.org/10.1042/bj3080261.
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The binding of the transport inhibitor, forskolin, to the galactose-H+ symporter, GalP, of Escherichia coli was evaluated by equilibrium and time-resolved fluorescence measurements. A quench in protein fluorescence of 8-12% was observed upon the binding of forskolin. The overall dissociation constant (Kd) for forskolin determined by fluorescence titration ranged between 1.2 and 2.2 microM, which is similar to that reported from equilibrium dialysis measurements of the binding of [3H]forskolin (Kd = 0.9-1.4 microM). The kinetics of forskolin binding were measured by stopped-flow fluorescence methods. The protein fluorescence was quenched in a biphasic manner; the faster of these two rates was dependent on the concentration of forskolin and was interpreted as the initial binding step from which both the association (kon) and dissociation (koff) rate constants were determined. The association and dissociation rate constants were 5.4-6.2 microM-1.s-1 and 5.1-11.5 s-1 respectively, and the Kd was calculated to be 1.5 microM. The binding of forskolin was inhibited by D-galactose, but not by L-galactose, and displacement by sugar provided an additional method to calculate the dissociation rate constant for forskolin (koff = 12.4-13.0 s-1). The rate of the slow change in protein fluorescence (3-5 s-1) was independent of the forskolin concentration, indicating an isomerization of the transporter between different conformations, possibly outward- and inward-facing forms. These kinetic parameters were determined at a series of temperatures, so that the thermodynamics of forskolin binding and transporter re-orientation could be analysed. The binding process was entropically driven (delta S = 83.7 J.K-1.mol-1; delta H = 8.25 kJ.mol-1), similar to that for cytochalasin B, which is also an inhibitor of GalP. Measurements of the binding of [3H]forskolin by equilibrium dialysis revealed competitive displacement of bound forskolin by cytochalasin B, possibly suggesting that the sugar, forskolin and cytochalasin B binding sites are overlapping; the Kds for forskolin and cytochalasin B were calculated to be 0.85 microM and 4.77 microM respectively, and the concentration of binding sites was 10.2 nmol.mg-1.
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Chen, C. K., T. J. McDonald, and E. E. Daniel. "Characterization of galanin receptor in canine small intestinal circular muscle synaptosomes." American Journal of Physiology-Gastrointestinal and Liver Physiology 266, no. 1 (January 1, 1994): G106—G112. http://dx.doi.org/10.1152/ajpgi.1994.266.1.g106.
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We used 125I-galanin (porcine) as ligand to study the galanin receptors in circular muscle and deep muscular plexus from canine small intestine. Specific binding sites were found in both nerve and muscle membranes. On synaptosomal membranes, the equilibrium binding study showed a high-affinity (dissociation constant, Kd = 1.1 +/- 0.13 nM; maximum binding capacity, Bmax = 244 +/- 2.1 fmol/mg) binding site. The specific binding of 125I-galanin to nerve membrane was inhibited by galanin or NH2-terminal galanin fragments but not by the COOH-terminal fragment. Computer analysis suggested a two-site model (inhibitor constants, Ki1 = 0.02 +/- 0.005 nM and Ki2 = 1.05 +/- 0.3 nM) for competition by galanin-(1-29). Kinetic and competition studies using guanosine 5'-O-(3-thiotriphosphate) or pertussis toxin (PTX) suggested that the high-affinity binding site involved a PTX-sensitive G protein which acted to slow dissociation of bound galanin from the receptor. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the galanin receptor complex revealed a radioactive band at 50 kDa. We conclude that, in canine small intestine, galanin may act as an inhibitory neuromodulator by a PTX-sensitive G protein-coupled interaction of galanin and its specific receptor on enteric nerve synaptosomes
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Raines, Douglas E., and Vinu T. Zachariah. "Isoflurane Increases the Apparent Agonist Affinity of the Nicotinic Acetylcholine Receptor by Reducing the Microscopic Agonist Dissociation Constant." Anesthesiology 92, no. 3 (March 1, 2000): 775–85. http://dx.doi.org/10.1097/00000542-200003000-00021.
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Background Isoflurane increases the apparent agonist affinity of ligand-gated ion channels. This action reflects a reduction in the receptor's agonist dissociation constant and/or the preopen/open channel state equilibrium. To evaluate the effect of isoflurane on each of these kinetic constants in the nicotinic acetylcholine receptor, the authors analyzed isoflurane's actions on (1) the binding of the fluorescent agonist Dns-C6-Cho to the nicotinic acetylcholine receptor's agonist self-inhibition site and (2) the desensitization kinetics induced by the binding of the weak partial agonist suberyldicholine. Methods The dissociation constant for Dns-C6-Cho binding to the self-inhibitory site was determined using stopped-flow fluorescence spectroscopy. The values of the kinetic constants for agonist binding, channel gating, and desensitization were determined by modeling the suberyldicholine concentration-dependence of the apparent rate of desensitization. Results Isoflurane did not significantly alter the dissociation constant for Dns-C6-Cho binding to the self-inhibitory site even at a concentration as high as 1.5 mM, the highest concentration studied. At this concentration, isoflurane substantially reduced the dissociation constant for suberyldicholine binding to its channel opening site by 97% from 17 +/- 5 microM to 0.5 +/- 0.2 microM, whereas the preopen/open channel state equilibrium was reduced only from 19.1 to 5 +/- 1. Conclusions Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor primarily by reducing the agonist dissociation constant of the site responsible for channel opening rather than altering channel gating kinetics.
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FRANSSEN, Jo, Irene SALEMINK, George M. WILLEMS, Tze-Chein WUN, H. Coenraad HEMKER, and Theo LINDHOUT. "Prothrombinase is protected from inactivation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor*." Biochemical Journal 323, no. 1 (April 1, 1997): 33–37. http://dx.doi.org/10.1042/bj3230033.
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The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin. The rate constant of association of prothrombinase with full-length TFPI was 2.1×107 M-1ċs-1 and 0.05×107 M-1ċs-1 for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65×10-4 s-1 in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.
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Zhu, Jingjing, Jeroen Declercq, Bart Roucourt, Gholamreza H. Ghassabeh, Sandra Meulemans, Jörg Kinne, Guido David, et al. "Generation and characterization of non-competitive furin-inhibiting nanobodies." Biochemical Journal 448, no. 1 (October 18, 2012): 73–82. http://dx.doi.org/10.1042/bj20120537.
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The PC (proprotein convertase) furin cleaves a large variety of proproteins and hence plays a major role in many pathologies. Therefore furin inhibition might be a good strategy for therapeutic intervention, and several furin inhibitors have been generated, although none are entirely furin-specific. To reduce potential side effects caused by cross-reactivity with other proteases, dromedary heavy-chain-derived nanobodies against catalytically active furin were developed as specific furin inhibitors. The nanobodies bound only to furin but not to other PCs. Upon overexpression in cell lines, they inhibited the cleavage of two different furin substrates, TGFβ (transforming growth factor β) and GPC3 (glypican 3). Purified nanobodies could inhibit the cleavage of diphtheria toxin into its enzymatically active A fragment, but did not inhibit cleavage of a small synthetic peptide-based substrate, suggesting a mode-of-action based on steric hindrance. The dissociation constant of purified nanobody 14 is in the nanomolar range. The nanobodies were non-competitive inhibitors with an inhibitory constant in the micromolar range as demonstrated by Dixon plot. Furthermore, anti-furin nanobodies could protect HEK (human embryonic kidney)-293T cells from diphtheria-toxin-induced cytotoxicity as efficiently as the PC inhibitor nona-D-arginine. In conclusion, these antibody-based single-domain nanobodies represent the first generation of highly specific non-competitive furin inhibitors.
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Stachyra, Thérèse, Marie-Claude Péchereau, Jean-Michel Bruneau, Monique Claudon, Jean-Marie Frère, Christine Miossec, Kenneth Coleman, and Michael T. Black. "Mechanistic Studies of the Inactivation of TEM-1 and P99 by NXL104, a Novel Non-β-Lactam β-Lactamase Inhibitor." Antimicrobial Agents and Chemotherapy 54, no. 12 (October 4, 2010): 5132–38. http://dx.doi.org/10.1128/aac.00568-10.
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ABSTRACT NXL104 is a potent inhibitor of class A and C serine β-lactamases, including KPC carbapenemases. Native and NXL104-inhibited TEM-1 and P99 β-lactamases analyzed by liquid chromatography-electrospray ionization-time of flight mass spectrometry revealed that the inactivated enzymes formed a covalent adduct with NXL104. The principal inhibitory characteristics of NXL104 against TEM-1 and P99 β-lactamases were determined, including partition ratios, dissociation constants (K), rate constants for deactivation (k 2), and reactivation rates. NXL104 is a potent inhibitor of TEM-1 and P99, characterized by high carbamylation efficiencies (k 2/K of 3.7 × 105 M−1 s−1 for TEM-1 and 1 × 104 M−1 s−1 for P99) and slow decarbamylation. Complete loss of β-lactamase activity was obtained at a 1/1 enzyme/NXL104 ratio, with a k 3 value (rate constant for formation of product and free enzyme) close to zero for TEM-1 and P99. Fifty percent inhibitory concentrations (IC50s) were evaluated on selected β-lactamases, and NXL104 was shown to be a very potent inhibitor of class A and C β-lactamases. IC50s obtained with NXL104 (from 3 nM to 170 nM) were globally comparable on the β-lactamases CTX-M-15 and SHV-4 with those obtained with the comparators (clavulanate, tazobactam, and sulbactam) but were far lower on TEM-1, KPC-2, P99, and AmpC than those of the comparators. In-depth studies on TEM-1 and P99 demonstrated that NXL104 had a comparable or better affinity and inactivation rate than clavulanate and tazobactam and in all cases an improved stability of the covalent enzyme/inhibitor complex.
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López, Mercedes, Goetz Nowak, and Thomas Bitter. "Design and characterization of dipetacompinR10H, a dipetalogastin II-derived, classical competitive thrombin inhibitor." Thrombosis and Haemostasis 97, no. 01 (2007): 139–45. http://dx.doi.org/10.1160/th06-06-0308.
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SummaryThe design of small chimeric thrombin inhibitors based on the structure of dipetalogastin II has been previously described. These proteins are effective inhibitors of thrombin showing slow binding or slow, tight-binding kinetics. We report here about dipetacompinR10H, a new dipetalogastin II-derived chimeric thrombin inhibitor, which exhibits classical competitive kinetics. The dissociation constant Ki of dipetacompinR10H was determined to be 17.1 ± 0.8 pM. In various coagulation assays it showed a comparable anticoagulant activity like r-hirudin and r-dipetalogastin II. DipetacompinR10H’s inhibition of thrombin was specific, since no inhibition of other serine proteases like factor Xa, plasmin, trypsin or chymotrypsin has been observed.
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Blessinger, K. J., and G. Tunnicliff. "Kinetics of inactivation of 4-aminobutyrate aminotransferase by 3-bromopyruvate." Biochemistry and Cell Biology 70, no. 8 (August 1, 1992): 716–19. http://dx.doi.org/10.1139/o92-109.
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3-Bromopyruvate inhibited 4-aminobutyrate aminotransferase (EC 2.6.1.19) from Pseudomonas fluorescens, apparently irreversibly. Kinetics of this inactivation were studied by continuously monitoring the enzyme reaction at 30 °C in the presence of inhibitor. Irrespective of how high an inhibitor concentration was present, a maximum rate of inactivation was eventually achieved (5.9 × 10−3 s−1), indicating the formation of a reversible inhibitor–enzyme complex before the final inactivation step. The dissociation constant of this complex was found to be 6.5 μM. This affinity labelling by 3-bromopyruvate suggests the presence of essential sulphydryl groups on the enzyme, since this compound is known to preferentially alkylate cysteinyl residues.Key words: 4-aminobutyrate, 4-aminobutyrate aminotransferase, inactivation, 3-bromopyruvate, affinity label, Pseudomonas fluorescens.
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Dong, Qi, Na Hu, Huilan Yue, and Honglun Wang. "Inhibitory Activity and Mechanism Investigation of Hypericin as a Novel α-Glucosidase Inhibitor." Molecules 26, no. 15 (July 28, 2021): 4566. http://dx.doi.org/10.3390/molecules26154566.
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α-glucosidase is a major enzyme that is involved in starch digestion and type 2 diabetes mellitus. In this study, the inhibition of hypericin by α-glucosidase and its mechanism were firstly investigated using enzyme kinetics analysis, real-time interaction analysis between hypericin and α-glucosidase by surface plasmon resonance (SPR), and molecular docking simulation. The results showed that hypericin was a high potential reversible and competitive α-glucosidase inhibitor, with a maximum half inhibitory concentration (IC50) of 4.66 ± 0.27 mg/L. The binding affinities of hypericin with α-glucosidase were assessed using an SPR detection system, which indicated that these were strong and fast, with balances dissociation constant (KD) values of 6.56 × 10−5 M and exhibited a slow dissociation reaction. Analysis by molecular docking further revealed that hydrophobic forces are generated by interactions between hypericin and amino acid residues Arg-315 and Tyr-316. In addition, hydrogen bonding occurred between hypericin and α-glucosidase amino acid residues Lys-156, Ser-157, Gly-160, Ser-240, His-280, Asp-242, and Asp-307. The structure and micro-environment of α-glucosidase enzymes were altered, which led to a decrease in α-glucosidase activity. This research identified that hypericin, an anthracene ketone compound, could be a novel α-glucosidase inhibitor and further applied to the development of potential anti-diabetic drugs.
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Gadosy, Timothy A., and Oswald S. Tee. "Spectator catalysis in the cleavage of p-nitrophenyl acetate and p-nitrophenyl hexanoate by "hydroxypropyl-β-cyclodextrin"." Canadian Journal of Chemistry 74, no. 5 (May 1, 1996): 745–52. http://dx.doi.org/10.1139/v96-081.
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Aliphatic alcohols that form host–guest complexes with "hydroxypropyl-β-cyclodextrin" retard the cleavage of m-nitrophenyl acetate by hydroxypropyl-β-cyclodextrin in basic aqueous solution, due to competitive inhibition. By contrast, these same species do not inhibit the reaction of p-nitrophenyl acetate and p-nitrophenyl hexanoate to the same extent and, in some cases, the addition of alcohols serves to increase the rate of reaction. The observed reaction kinetics require the presence of a process that has one molecule of the "potential inhibitor" in the transition state for ester cleavage. Rate constants, ka, for the reaction of the {ester•hydroxypropyl-β-cyclodextrin} complexes with a series of potential inhibitors show a strong dependence on the ability of the potential inhibitor to bind to the cyclodextrin. On the other hand, rate constants for the kinetically equivalent reaction of the ester with the {cyclodextrin•potential inhibitor} complex show little dependence on the alcohol structure and they vary over a very limited range. The negative logarithms of the apparent dissociation constant of the potential inhibitor from the transition state show a strong dependence on the ability of the potential inhibitor to bind to hydroxypropyl-β-cyclodextrin, indicating that the binding of the potential inhibitor in the initial state and the transition state is similar. It is concluded that the cleavage of p-nitrophenyl acetate and p-nitrophenyl hexanoate by hydroxypropyl-β-cyclodextrin in the presence of 14 potential inhibitors can occur with the ester largely outside of the hydroxypropyl-β-cyclodextrin cavity during the transition state, allowing the cavity to be occupied by a molecule of potential inhibitor. Key words: cyclodextrin, spectator catalysis, esterolysis.
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Inglese, James, Richard A. Blatchly, and Stephen J. Benkovic. "A multisubstrate adduct inhibitor of a purine biosynthetic enzyme with a picomolar dissociation constant." Journal of Medicinal Chemistry 32, no. 5 (May 1989): 937–40. http://dx.doi.org/10.1021/jm00125a002.
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Yamanishi, R., J. Kotera, T. Fushiki, T. Soneda, T. Saitoh, T. Oomori, T. Satoh, and E. Sugimoto. "A specific binding of the cholecystokinin-releasing peptide (monitor peptide) to isolated rat small-intestinal cells." Biochemical Journal 291, no. 1 (April 1, 1993): 57–63. http://dx.doi.org/10.1042/bj2910057.
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A specific binding of the cholecystokinin (CCK)-releasing peptide (monitor peptide) to isolated rat jejunal mucosal cells was investigated. The 125I-labelled purified monitor peptide bound to the rat jejunal cells, and a large excess amount of the non-labelled monitor peptide inhibited the binding. The binding was completed within 60 min at 37 degrees C. The optimum pH for the binding was 8-9. A Scatchard plot of the specific binding was linear, and the dissociation constant was 50 nM. The density of the monitor-peptide-binding sites was high in duodenum but low in ileal and absent in colonic mucosa. A recombinant monitor peptide and four kinds of point mutants of it were prepared. The binding of the mutant monitor peptides to the cells indicated that only a trypsin inhibitor of the mutants could bind to the mucosal cells. Human pancreatic secretory trypsin inhibitor inhibited the specific binding, but other trypsin inhibitors, i.e. bovine basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, egg-white trypsin inhibitor, leupeptin, antipain and FOY-305, did not affect the specific binding at all. These findings suggested that the specific binding site for the monitor peptide on the jejunal mucosal cells has a trypsin-like specificity, exhibiting a special specificity for the pancreatic-secretory-trypsin-inhibitor family. Autoradiography of an affinity-cross-linked complex of the 125I-labelled intact monitor peptide and the binding site suggested that its molecular mass was 33 kDa or 53 kDa in the presence or absence of 2-mercaptoethanol respectively.
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Liu, Yaqin, Yuanjiang Pan, and Yuhong Xu. "Binding Investigation of Integrin αvβ3 With Its Inhibitors by SPR Technology and Molecular Docking Simulation." Journal of Biomolecular Screening 15, no. 2 (January 19, 2010): 131–37. http://dx.doi.org/10.1177/1087057109356207.
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Integrins play critical roles in the process of angiogenesis and are attractive targets for anticancer therapies. It is desirable to develop new types of small-molecule inhibitors of integrin. Herein, the binding features of several inhibitors to integrin αvβ3 have been studied by surface plasmon resonance (SPR) biosensor technology and molecular docking analyses. The SPR results indicated that the equilibrium dissociation constant (KD) values are evaluated for the inhibitors and showed that the KD value of cyclopeptide c-Lys is much lower than the reference molecule. In addition, the 3D structural model of integrin αvβ3 was generated according to the crystal structure of the integrin αvβ3 complex, and the molecular docking simulation analyses revealed that the predicted binding sites for the most active cyclopeptide c-Lys were consistent with the reported structure. These results thus implied that cyclopeptide c-Lys could be developed as a novel inhibitor for integrin αvβ3. The current work has potential for application in structure-based integrin αvβ3 inhibitor discovery.
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Mock, W. L., D. J. Freeman, and M. Aksamawati. "Fluxionate Lewis acidity of the Zn2+ ion in carboxypeptidase A." Biochemical Journal 289, no. 1 (January 1, 1993): 185–93. http://dx.doi.org/10.1042/bj2890185.
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Competitive inhibition constants Ki for a series of phenol-ring-substituted derivatives of alpha-(2-hydroxyphenyl)benzenepropanoic acid have been ascertained by observing their influence on the catalytic hydrolysis of a peptide substrate by the zinc enzyme carboxypeptidase A. The pH-dependence of Ki shows that binding is maximal between two pKa values: one is that of the phenol group of the inhibitor, and the other uniformly has a value of 6, the pKa of a Zn(2+)-bound water molecule on the enzyme in the absence of substrate or inhibitor. This is the dependence expected if phenolate binds to the Zn2+ displacing its bound H2O/HO-. A log-log plot of the dissociation constants for the productive forms of inhibitor plus enzyme versus the acid dissociation constants of the phenolic residues in the inhibitors yields a straight line with a slope of +0.76. This number indicates that the active-site metal ion has special capacity for dispersing negative charge, such as builds up on the oxygen atom of a carboxamide group undergoing nucleophilic addition.
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Chapman, H. A., and O. L. Stone. "Characterization of a macrophage-derived plasminogen-activator inhibitor. Similarities with placental urokinase inhibitor." Biochemical Journal 230, no. 1 (August 15, 1985): 109–16. http://dx.doi.org/10.1042/bj2300109.
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Human and mouse macrophages release a fibrinolytic inhibitor after stimulation by endotoxin in vitro. The released mouse inhibitor was indistinguishable in size by molecular-sieve chromatography from an intracellular form (approx. 50 kDa), and both inhibitors blocked urokinase directly as judged by a 125I-plasminogen conversion assay. The intracellular inhibitor was found mostly to dissociate from 125I-urokinase during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reduced conditions, but a dodecyl sulphate-stable complex at 65-67 kDa was observed. Because of similarities in the reported size, stability and urokinase-binding properties of a placental urokinase inhibitor, the kinetic properties of the two inhibitors were compared. Under the reaction conditions employed (37 degrees C at pH7.4 in the presence of 0.2% Triton X-100), the association rate constants and equilibrium dissociation constants of the two inhibitors were indistinguishable, 3 × 10(5) M-1 × s-1 and 4 × 10(-10) M respectively. These data show that peritoneal macrophages contain a plasminogen-activator very similar to a previously recognized placental inhibitor. Although the inhibitor appears to be a trace protein in macrophages, placental macrophages may account for the accumulation of the inhibitor in placental tissue.
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Liu, S. Q., E. Ries, and P. A. Knauf. "Effects of external pH on binding of external sulfate, 4.4-dinitro-stilbene-2,2'-disulfonate (DNDS), and chloride to the band 3 anion exchange protein." Journal of General Physiology 107, no. 2 (February 1, 1996): 293–306. http://dx.doi.org/10.1085/jgp.107.2.293.
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A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.
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Faller, B., S. Dirrig, M. Rabaud, and J. G. Bieth. "Kinetics of the inhibition of human pancreatic elastase by recombinant eglin c. Influence of elastin." Biochemical Journal 270, no. 3 (September 15, 1990): 639–44. http://dx.doi.org/10.1042/bj2700639.
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Recombinant eglin c is a potent reversible inhibitor of human pancreatic elastase. At pH 7.4 and 25 degrees C, kass. = 7.3 x 10(5) M-1.s-1, kdiss. = 2.7 x 10(-4) s-1 and Ki = 3.7 x 10(-10) M. Stopped-flow kinetic indicate that the formation of the stable enzyme-inhibitor complex is not preceded by a fast pre-equilibrium complex or that the latter has a dissociation constant greater than 0.3 microM. The elastase-eglin c complex is much less stable at pH 5.0 and 25 degrees C, where kdiss. = 1.1 x 10(-2) s-1 and Ki = 7.3 x 10(-8) M. At pH 7.4 the activation energy for kass. is 43.9 kJ.mol-1 (10.5 kcal.mol-1). The kass. increases between pH 5.0 and 8.0 and remains essentially constant up to pH 9.0. This pH-dependence could not be described by a simple ionization curve. Both alpha 2-macroglobulin and alpha 1-proteinase inhibitor are able to dissociate the elastase-eglin c complex, as evidenced by measurement of the enzymic activity of alpha 2-macroglobulin-bound elastase or by polyacrylamide-gel electrophoresis of mixtures of alpha 1-proteinase inhibitor and elastase-eglin c complex. The rough estimate of kdiss. obtained with the alpha 2-macroglobulin dissociation experiment (1.6 x 10(-4) s-1) was of the same order of magnitude as the constant measured with the progress curve method. Eglin c strongly inhibits the solubilization of human aorta elastin by human pancreatic elastase. The extent of inhibition is the same whether elastase is added to a suspension of elastin and eglin c or whether elastase is preincubated with elastin for 3 min before addition of eglin c. However, the efficiency of the inhibitor sharply decreases if elastase is reacted with elastin for more prolonged periods.
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Yoshino, M. "A graphical method for determining inhibition parameters for partial and complete inhibitors." Biochemical Journal 248, no. 3 (December 15, 1987): 815–20. http://dx.doi.org/10.1042/bj2480815.
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A new simple graphical method is described for the determination of inhibition type and kinetic parameters of an enzyme reaction without any replot. The method consists of plotting experimental data as v/(vo-v) versus the reciprocal of the inhibitor concentration at different substrate concentrations, where v and vo represent the velocity in the presence and in the absence of the inhibitor respectively with a given concentration of the substrate. Partial inhibition gives straight lines that converge on the abscissa at a point away from the origin, whereas complete inhibition gives lines that go through the origin. The inhibition constants of enzymes and the reaction rate constant of the enzyme-substrate-inhibitor complex can be calculated from the abscissa and ordinate intercepts of the plot. The relationship between the slope of the plot and the substrate concentration shows characteristic features depending on the inhibition type: for partial competitive inhibition, the straight line converging on the abscissa at-Ks, the dissociation constant of the enzyme-substrate complex; for non-competitive inhibition, a constant slope independent of the substrate concentration; for uncompetitive inhibition, a hyperbola decreasing with the increase in the substrate concentration; for mixed-type inhibition, a hyperbola increasing with the increase in the substrate concentration. The properties of the replot are useful in confirmation of the inhibition mechanism.
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Williams, H. R., T. Y. Lin, M. A. Navia, J. P. Springer, and K. Hoogsteen. "Pig pancreatic anhydro-elastase. Role of the serine-195 hydroxy group in the binding of inhibitors and substrate." Biochemical Journal 242, no. 1 (February 15, 1987): 267–73. http://dx.doi.org/10.1042/bj2420267.
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The binding constants of a number of ligands were measured for pancreatic elastase (PE) and anhydro-elastase (AE) in order to assess the contribution of Ser-195 to substrate and inhibitor binding by PE. AE was purified by affinity chromatography on a column containing immobilized turkey ovomucoid inhibitor. The AE had 0.1 +/- 0.1% of the activity of the native enzyme and contained 0.8 +/- 0.06 residue of dehydroalanine per molecule. A difference electron-density map, derived from an X-ray crystallographic analysis of AE, showed that the modified residue was Ser-195. The complexing of 3-carboxypropionyl-Ala-Ala-Ala-p-nitroanilide (SAN) to the active site of AE was also demonstrated by X-ray-diffraction analysis of an AE crystal soaked overnight with substrate. The nitroanilide moiety was not observed in the difference map. AE was shown to bind turkey ovomucoid inhibitor with a dissociation constant (Kd) of 0.3 +/- 0.06 microM compared with 0.10 microM for PE. The Kd of the AE-SAN complex (0.2 mM) was comparable with the Michaelis constant for SAN with PE (1.0 mM). A number of inhibitors, such as elastatinal, which forms a hemiketal adduct with PE, while others such as the beta-lactams, which function as acylators of the active-site serine residue, bound AE with a lower affinity than to PE. The binding of a peptidylchloromethane (acetyl-Ala-Ala-Pro-Ala-CH2Cl) to AE occurs without evidence for alkylation of histidine. The binding constants for benzoisothiazolinone and 3,4-dichloroisocoumarin to PE differed from their binding constants to AE by less than a factor of 4.0-fold. The contribution of the hydroxy group of Ser-195 to the binding of these inhibitors to PE in their non-covalent complexes is relatively small, even though they inactivate PE by an acylation mechanism. These results suggest that the hydroxy group on Ser-195 in PE is of secondary importance in the energetics of ligand binding, in contrast with its essential role in the catalytic properties of the enzyme.
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Volpe, P., B. H. Alderson-Lang, and G. A. Nickols. "Regulation of inositol 1,4,5-trisphosphate-induced Ca2+ release. I. Effect of Mg2+." American Journal of Physiology-Cell Physiology 258, no. 6 (June 1, 1990): C1077—C1085. http://dx.doi.org/10.1152/ajpcell.1990.258.6.c1077.
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Canine cerebellar membranes were fractionated by differential centrifugation into a crude mitochondrial pellet (P2) and a crude microsomal pellet (P3). The effect of Mg2+ on inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release and [3H]IP3 binding was assessed. Mg2+ inhibited IP3-induced Ca2+ release in a concentration-dependent manner. Mg2+ influenced both the extent of IP3-induced Ca2+ release and the apparent affinity for IP3. A 10-fold change of free Mg2+ (from approximately 30 to approximately 300 microM) reduced the extent of Ca2+ release by two- to threefold and shifted the apparent Michaelis constant from approximately 0.5 to approximately 0.9 microM IP3. Thus Mg2+ seemed to be noncompetitive inhibitor of IP3-induced Ca2+ release. Mg2+ also inhibited Ca2+ release elicited by glycerophosphoinositol 4,5-bisphosphate, a poorly metabolized analogue of IP3. Mg2+ and heparin sodium were shown to be additive inhibitors of IP3-induced Ca2+ release. Mg2+ inhibited [3H]IP3 binding under experimental conditions designed to minimize IP3 hydrolysis. Scatchard plots indicated that 0.5 mM free Mg2+ reduced maximum binding from 10.9 to 3.5 pmol IP3 bound/mg protein and increased the dissociation constant from 136 to 227 nM. The modulation of [3H]IP3 binding and IP3-induced Ca2+ release by Mg2+ could be physiologically relevant.
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Nazir, Yasir, Hummera Rafique, Sadia Roshan, Shazia Shamas, Zaman Ashraf, Muhammad Rafiq, Tehreem Tahir, Zia-Ur-Rahman Qureshi, Alvina Aslam, and Muhammad Hassham Hassan Bin Asad. "Molecular Docking, Synthesis, and Tyrosinase Inhibition Activity of Acetophenone Amide: Potential Inhibitor of Melanogenesis." BioMed Research International 2022 (January 11, 2022): 1–10. http://dx.doi.org/10.1155/2022/1040693.
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Tyrosinase and its related proteins are responsible for pigmentation disorders, and inhibiting tyrosinase is an established strategy to treat hyperpigmentation. The carbonyl scaffolds can be effective inhibitors of tyrosinase activity, and the fact that both benzoic and cinnamic acids are safe natural substances with such a scaffolded structure, it was speculated that hydroxyl-substituted benzoic and cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. These moieties were incorporated into new chemotypes that displayed in vitro inhibitory effect against mushroom tyrosinase with a view to explore antimelanogenic ingredients. The most active compound, 2-((3-acetylphenyl)amino)-2-oxoethyl(E)-3-(2,4-dihydroxyphenyl)acrylate (5c), inhibited mushroom tyrosinase with an IC50 of 0.0020 ± 0.0002 μ M , while 2-((3-acetylphenyl)amino)-2-oxoethyl 2,4-dihydroxybenzoate (3c) had an IC50 of 27.35 ± 3.6 μ M in comparison to the positive control arbutin and kojic acid with a tyrosinase inhibitory activity of IC50 of 191.17 ± 5.5 μ M and IC50 of 16.69 ± 2.8 μ M , respectively. Analysis of enzyme kinetics revealed that 5c is a competitive and reversible inhibitor with dissociation constant (Ki) value 0.0072 μM. In silico docking studies with mushroom tyrosinase (PDB ID 2Y9X) predicted possible binding modes in the enzymatic pocket for these compounds. The orthohydroxyl of the cinnamic acid moiety of 5c is predicted to form hydrogen bond with the active site side chain carbonyl of Asn 260 (2.16 Å) closer to the catalytic site Cu ions. The acetyl carbonyl is picking up another hydrogen bond with Asn 81 (1.90 Å). The inhibitor 5c passed the panassay interference (PAINS) alerts. This study presents the potential of hydroxyl-substituted benzoic and cinnamic acids and could be beneficial for various cosmetic formulations.
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Crawford, Jeremie J., Joshua W. Hollett, and Douglas B. Craig. "Determination of the inhibitor dissociation constant of an individual unmodified enzyme molecule in free solution." ELECTROPHORESIS 37, no. 15-16 (June 29, 2016): 2217–25. http://dx.doi.org/10.1002/elps.201600201.
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Chen, C. K., T. J. McDonald, and E. E. Daniel. "Galanin receptor in plasma membrane of canine small intestinal circular muscle." American Journal of Physiology-Gastrointestinal and Liver Physiology 266, no. 1 (January 1, 1994): G113—G117. http://dx.doi.org/10.1152/ajpgi.1994.266.1.g113.
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High-affinity binding sites for galanin were identified and characterized in plasma membrane of circular muscle from canine small intestine using 125I-radioiodinated synthetic porcine galanin. Scatchard analysis indicated a high-affinity binding site on plasma membrane with a dissociation constant (Kd) of 0.58 nM and a binding capacity of 389 fmol/mg. Unlabeled galanin or NH2-terminal galanin fragments competitively inhibited the binding of 125I-galanin in a concentration-dependent manner, whereas the COOH-terminal fragment was inactive. Computer analysis of competitive binding data suggested a two-site model with a high-affinity (inhibitor constant, Ki = 0.01 nM) and a low-affinity (Ki = 2.8 nM) binding site. Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) enhanced the dissociation of bound 125I-galanin. Cholera toxin (CTX) and GTP gamma S abolished the activity of the high-affinity binding site, leaving the low-affinity binding site. We conclude that galanin may act as an neurotransmitter to inhibit canine small intestinal smooth muscle contraction by interaction with a CTX-sensitive G protein-coupled specific receptor on muscle membrane. This receptor showed different G protein coupling from a synaptosomal receptor previously described in the same tissue preparations
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Takai, A., Y. Ohno, T. Yasumoto, and G. Mieskes. "Estimation of the rate constants associated with the inhibitory effect of okadaic acid on type 2A protein phosphatase by time-course analysis." Biochemical Journal 287, no. 1 (October 1, 1992): 101–6. http://dx.doi.org/10.1042/bj2870101.
Full textAbstract:
As is often the case with tightly binding inhibitors, okadaic acid produces its inhibitory effect on type 2A protein phosphatase (PP2A) in a time-dependent manner. We measured the rate constants associated with the binding of okadaic acid to PP2A by analysing the time-course of the reduction of the p-nitrophenyl phosphate (pNPP) phosphatase activity of the enzyme after application of okadaic acid. The rate constants for dissociation of okadaic acid from PP2A were also estimated from the time-course of the recovery of the activity from inhibition by okadaic acid after addition of a mouse IgG1 monoclonal antibody raised against the inhibitor. Our results show that the rate constants for the binding of okadaic acid and PP2A are of the order of 10(7) M-1.s-1, a typical value for reactions involving relatively large molecules, whereas those for their dissociation are in the range 10(-4)-10(-3) s-1. The very low values of the latter seems to be the determining factor for the exceedingly high affinity of okadaic acid for PP2A. The dissociation constants for the interaction of okadaic acid with the free enzyme and the enzyme-substrate complex, estimated as the ratio of the rate constants, are both in the range 30-40 pM, in agreement with the results of previous dose-inhibition analyses.
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Wierzchowski, Jacek, Agnieszka Bzowska, Katarzyna Stępniak, and David Shugar. "Interactions of Calf Spleen Purine Nucleoside Phosphorylase with 8-Azaguanine, and a Bisubstrate Analogue Inhibitor: Implications for the Reaction Mechanism." Zeitschrift für Naturforschung C 59, no. 9-10 (October 1, 2004): 713–25. http://dx.doi.org/10.1515/znc-2004-9-1017.
Full textAbstract:
Abstract Interactions of calf spleen purine nucleoside phosphorylase (PNP) with a non-typical substrate, 8-azaguanine (8-azaG), and a bisubstrate analogue inhibitor, 9-(2-phosphonylmethoxyethyl)- 8-azaguanine (PME-azaG), were investigated by means of steady-state fluorescence spectroscopy. Both 8-azaG and PME-azaG form fluorescent complexes with the enzyme, and dissociation constants are comparable to the appropriate parameters (Km or Ki) obtained from kinetic measurements. PME-azaG inhibits both the phosphorolytic and synthetic pathway of the reaction in a competitive mode. The complex of 8-azaG with PNP is much weaker than the previously reported Gua-PNP complex, and its dissociation constant increases at pH > 7, where 8-azaG exists predominantly as the monoanion (pKa ≈ 6.5). The fluorescence difference spectrum of the PNP/8-azaG complex points to participation of the N(7)H or/and N(8)H tautomers of the neutral substrate, and the 9-(2-phosphonylmethoxyethyl) derivative also exists as a neutral species in the complex with PNP. The latter conclusion is based on spectral characteristics of the PNP/PME-azaG complex, confirmed by fluorimetric determination of dissociation constants, which are virtually pH-independent in the range 6-7. These findings testify to involvement of the neutral purine molecule, and not its monoanion, as the substrate in the reverse, synthetic reaction. It is proposed that, in the reverse reaction pathway, the natural purine substrate is bound to the enzyme as the neutral N(7)H tautomer, which is responsible for the reported strong fluorescence of the guanine-PNP complex.
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Zaborska, W., M. Leszko, M. Kot, and A. Juszkiewicz. "The enthalpimetric determination of inhibition constants for the inhibition of urease by acetohydroxamic acid." Acta Biochimica Polonica 44, no. 1 (March 31, 1997): 89–98. http://dx.doi.org/10.18388/abp.1997_4444.
Full textAbstract:
The effect of concentration of acetohydroxamic acid (AHA) on inhibition of jack bean urease in phosphate buffer, pH 7.0, at 25 degrees C, was studied. The measurements were performed at urease concentration of 2.5 mg/100 cm3 for concentrations of urea and AHA ranging in the range of 2-50 mmol dm-3 and 0.25-10 mmol dm-3, respectively. The reactions were monitored by two techniques: analytical and enthalpimetric. For the analytical technique the growth of ammonia concentration in the course of the reaction was determined. From the recorded progress curves the following parameters were calculated for each inhibitor concentration: the initial reaction rate, the steady-state rate and the inversion constant. From these parameters the inhibition constants of the initial and steady-state stages of the reaction, Ki and Ki, were calculated. The former constant did not change whereas the latter one proved to decrease quickly with an increase in inhibitor concentration. This behaviour resulted from the fact that the inactive complex EI was not a product of internal inversion but was formed in the reaction: 2/3I + EI-->(EI.2/3I). The dissociation constant of this complex is equal to about 0.3 x 10(-3) (mol dm-3)2/3.
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ALDERTON, Wendy K., Angela BOYHAN, and Peter N. LOWE. "Nitroarginine and tetrahydrobiopterin binding to the haem domain of neuronal nitric oxide synthase using a scintillation proximity assay." Biochemical Journal 332, no. 1 (May 15, 1998): 195–201. http://dx.doi.org/10.1042/bj3320195.
Full textAbstract:
Nitric oxide synthases (NOS) have a bidomain structure comprised of an N-terminal oxygenase domain and a C-terminal reductase domain. The oxygenase domain binds haem, (6R)-5,6,7,8-tetrahydro-l-biopterin (tetrahydrobiopterin) and arginine, is the site where nitric oxide synthesis takes place and contains determinants for dimeric interactions. A novel scintillation proximity assay has been established for equilibrium and kinetic measurements of substrate, inhibitor and cofactor binding to a recombinant N-terminal haem-binding domain of rat neuronal NOS (nNOS). Apparent Kd values for nNOS haem-domain-binding of arginine and Nω-nitro-l-arginine (nitroarginine) were measured as 1.6 µM and 25 nM respectively. The kinetics of [3H]nitroarginine binding and dissociation yielded an association rate constant of 1.3×104 s-1·M-1 and a dissociation rate constant of 1.2×10-4 s-1. These values are comparable to literature values obtained for full-length nNOS, suggesting that many characteristics of the arginine binding site of NOS are conserved in the haem-binding domain. Additionally, apparent Kd values were compared and were found to be similar for the inhibitors, l-NG-monomethylarginine, S-ethylisothiourea, N-iminoethyl-l-ornithine, imidazole, 7-nitroindazole and 1400W (N-[3-(aminomethyl) benzyl] acetamidine). [3H]Tetrahydrobiopterin bound to the nNOS haem domain with an apparent Kd of 20 nM. Binding was inhibited by 7-nitroindazole and stimulated by S-ethylisothiourea. The kinetics of interaction with tetrahydrobiopterin were complex, showing a triphasic binding process and a single off rate. An alternating catalytic site mechanism for NOS is proposed.
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Saboury, A. A., A. Divsalar, G. Ataie, M. Amanlou, A. A. Moosavi-Movahedi, and G. H. Hakimelahi. "Inhibition study of adenosine deaminase by caffeine using spectroscopy and isothermal titration calorimetry." Acta Biochimica Polonica 50, no. 3 (September 30, 2003): 849–55. http://dx.doi.org/10.18388/abp.2003_3676.
Full textAbstract:
Kinetic and thermodynamic studies were made on the effect of caffeine on the activity of adenosine deaminase in 50 mM sodium phosphate buffer, pH 7.5, using UV spectrophotometry and isothermal titration calorimetry (ITC). An uncompetitive inhibition was observed for caffeine. A graphical fitting method was used for determination of binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 350 microM by the microcalorimetry method, which agrees well with the value of 342 microM for the inhibition constant that was obtained from the spectroscopy method. Positive dependence of caffeine binding on temperature indicates a hydrophobic interaction.
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Capitani, Guido, Darla L. McCarthy, Heinz Gut, Markus G. Grütter, and Jack F. Kirsch. "Apple 1-Aminocyclopropane-1-carboxylate Synthase in Complex with the Inhibitor l-Aminoethoxyvinylglycine." Journal of Biological Chemistry 277, no. 51 (September 11, 2002): 49735–42. http://dx.doi.org/10.1074/jbc.m208427200.
Full textAbstract:
The 1.6-Å crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitorl-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 × 105m−1s−1) and by the rate of loss of enzyme activity (1.1 × 105m−1s−1). The dissociation rate constant determined by activity recovery is 2.4 × 10−6s−1. Thus, the calculatedKdvalue is 10–20 pm.
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Perilli, Mariagrazia, Alisia Mancini, Giuseppe Celenza, Carlo Bottoni, Pierangelo Bellio, Alessia Sabatini, Letizia Di Pietro, Fabrizia Brisdelli, Bernardetta Segatore, and Gianfranco Amicosante. "Kinetic Study of the Effect of Histidines 240 and 164 on TEM-149 Enzyme Probed by β-Lactam Inhibitors." Antimicrobial Agents and Chemotherapy 58, no. 10 (August 4, 2014): 6294–96. http://dx.doi.org/10.1128/aac.02950-14.
Full textAbstract:
ABSTRACTIn the present study, we performed a detailed kinetic analysis of the enzymes TEM-149, TEM-149H240, and TEM-149H164-H240versus a large panel of inhibitors/inactivators, including penicillins, penems, carbapenems, monobactams, cephamycin, and carbacephem. These compounds behaved as poor substrates versus TEM-149, TEM-149H240, and TEM-149H164-H240β-lactamases, and theKi(inhibition constant),K(dissociation constant of the Henri-Michaelis complex),k+2andk+3(first-order acylation and deacylation constants, respectively), andk+2/Kvalues were calculated.
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Maqtari, Maher Ali, and A. B. Mohamed Saad. "Screening and Purification of a Chymotrypsin Inhibitor from Entrolobium Saman Seeds." Sultan Qaboos University Journal for Science [SQUJS] 15 (December 1, 2010): 19. http://dx.doi.org/10.24200/squjs.vol15iss0pp19-29.
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A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAEcellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme- inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α -amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2 groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10o C-97o C.
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Kanalas, J. J. "Characterization of plasminogen system on rat yolk sac carcinoma (L2) cells." American Journal of Physiology-Cell Physiology 268, no. 2 (February 1, 1995): C442—C448. http://dx.doi.org/10.1152/ajpcell.1995.268.2.c442.
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The plasminogen (Plg) system on rat yolk sac carcinoma (L2) cells was characterized by zymography, Western and immunoprecipitation analysis, reverse-transcriptase polymerase chain reaction, binding, and activity assays. The L2 cells produced tissue Plg activator but not urokinase Plg activator and contained RNA for Plg activator inhibitor 1, but Plg activator inhibitor 1 was not detectable by zymography or Western analysis and contained the receptor for urokinase Plg activator. Plg bound to the cells in a saturable manner when plasmin inhibitors were present with a dissociation constant of 1.34 +/- 0.18 x 10(-6) M and 1.54 +/- 0.25 x 10(7) sites/cell. Immunoprecipitation analysis showed that Plg was binding to gp330, a known Plg receptor. Once bound to the L2 cells, Plg was activated by tissue Plg activator to plasmin in a time- and concentration-dependent manner. Under saturating Plg conditions, most of the plasmin produced was released into the medium. Inhibition of plasmin activation occurred when Plg activator inhibitor 1, anticatalytic tissue Plg activator antibody, or Heymann nephritis autoantibody was present.
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Nycander, M., and I. Björk. "Evidence by chemical modification that tryptophan-104 of the cysteine-proteinase inhibitor chicken cystatin is located in or near the proteinase-binding site." Biochemical Journal 271, no. 1 (October 1, 1990): 281–84. http://dx.doi.org/10.1042/bj2710281.
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The single tryptophan residue Trp-104 of chicken cystatin was modified with a 2-hydroxy-5-nitrobenzyl group. The change of the absorption spectrum of this group on binding of the modified cystatin to papain indicated a decreased environmental polarity of the probe. The modified inhibitor had about a 10(5)-fold lower affinity for papain than had intact cystatin, this being due to a higher dissociation rate constant. These results show that Trp-104 of cystatin is located in or near the proteinase-binding site of the inhibitor, in agreement with a model proposed from computer docking Experiments.
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Stratmann, Bernd, Martin Farr, and Harald Tschesche. "Characterization of C-Terminally Truncated Human Tissue Inhibitor of Metalloproteinases-4 Expressed in Pichia pastoris." Biological Chemistry 382, no. 6 (June 27, 2001): 987–91. http://dx.doi.org/10.1515/bc.2001.124.
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Abstract The tight regulation of extracellular matrix remodeling and degradation is of great importance in physiological processes like development and morphogenesis, as well as in pathological situations like tumor invasion and metastasis. Tissue inhibitors of metalloproteinases (TIMPs) are the naturally occuring inhibitors of matrix metalloproteinases, which are involved in matrix turnover. In this report we describe the cloning of human TIMP-4 from a human adenocarcinoma and an osteosarcoma cell line and the expression of the inhibitory domain in the methylotrophic yeast Pichia pastoris. The inhibition of MMP-8, -9, -12, -13 and -14 by the Nterminal domain of TIMP-4 was analysed. Using a fluorescent MCApeptide, K values for each subclass of MMPs were determined. With dissociation constants in the nanomolar range, TIMP-4 seems to be a good inhibitor for all classes of MMPs without remarkable preference for special MMPs.
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Prystay, Linda, Mylene Gosselin, and Peter Banks. "Determination of Equilibrium Dissociation Constants in Fluorescence Polarization." Journal of Biomolecular Screening 6, no. 3 (June 2001): 141–50. http://dx.doi.org/10.1177/108705710100600304.
Full textAbstract:
A simple mathematical model (the FP Kd model) is used to generate the dissociation equilibrium constant (Kd) for G protein-coupled receptor-ligand binding measured using fluorescence polarization (FP) saturation curve analysis. The model generates data that may be analyzed by the method of Scatchard. The validity of the FP Kd model is proven in six model systems in which the modeled Kd values are within a factor of 5 of inhibitory equilibrium constant values obtained from radioligand competition assays.